Immune-enhancing neutrophils reprogrammed by subclinical low-dose endotoxin in cancer treatment.

Despite the re-emergence of the pioneering "Coley's toxin" concept in anti-cancer immune therapies highlighted by check-point inhibitors and CAR-T approaches, fundamental mechanisms responsible for the immune-enhancing efficacy of low-dose "Coley's toxin" remain poorly understood. This study examines the novel reprogramming of immune-enhancing neutrophils by super-low dose endotoxin conducive for anti-cancer therapies. Through integrated analyses including scRNAseq and functional characterizations, we examined the efficacy of reprogrammed neutrophils in treating experimental cancer. We observed that neutrophils trained by super-low dose endotoxin adopt a potent immune-enhancing phenotype characterized by CD177loCD11bloCD80hiCD40hiDectin2hi. Both murine and human neutrophils trained by super-low dose endotoxin exhibit relieved suppression of adaptive T cells as compared to un-trained neutrophils. Functionally, neutrophils trained by super-low dose endotoxin can potently reduce tumor burden when transfused into recipient tumor-bearing mice. Mechanistically, Super-low dose endotoxin enables the generation of immune-enhancing neutrophils through activating STAT5 and reducing innate suppressor IRAK-M. Together, our data clarify the long-held mystery of "Coley's toxin" in rejuvenating anti-tumor immune defense, and provide a proof-of-concept in developing innate neutrophil-based anti-tumor therapeutics.

Assessing Endoscopic Response in Locally Advanced Rectal Cancer Treated with Total Neoadjuvant Therapy: Development and Validation of a Highly Accurate Convolutional Neural Network.

BACKGROUND: Rectal tumors display varying degrees of response to total neoadjuvant therapy (TNT). We evaluated the performance of a convolutional neural network (CNN) in interpreting endoscopic images of either a non-complete response to TNT or local regrowth during watch-and-wait surveillance. METHODS: Endoscopic images from stage II/III rectal cancers treated with TNT from 2012 to 2020 at a single institution were retrospectively reviewed. Images were labelled as Tumor or No Tumor based on endoscopy timing (before, during, or after treatment) and the tumor's endoluminal response. A CNN was trained using ResNet-50 architecture. The area under the curve (AUC) was analyzed during training and for two test sets. The main test set included images of tumors treated with TNT. The other contained images of local regrowth. The model's performance was compared to sixteen surgeons and surgical trainees who evaluated 119 images for evidence of tumor. Fleiss' kappa was calculated by respondent experience level. RESULTS: A total of 2717 images from 288 patients were included; 1407 (51.8%) contained tumor. The AUC was 0.99, 0.98, and 0.92 for training, main test, and local regrowth test sets. The model performed on par with surgeons of all experience levels for the main test set. Interobserver agreement was good ( k = 0.71-0.81). All groups outperformed the model in identifying tumor from images of local regrowth. Interobserver agreement was fair to moderate ( k = 0.24-0.52). CONCLUSIONS: A highly accurate CNN matched the performance of colorectal surgeons in identifying a noncomplete response to TNT. However, the model demonstrated suboptimal accuracy when analyzing images of local regrowth.

Practicing Surgeons' Perception of Barriers to Palliative Care Delivery in British Columbia.

BACKGROUND: Utilization of palliative care remains low among surgical patients. We aim to characterize general surgeons' perceptions of barriers to access palliative care in British Columbia (BC). METHODS: Semi-structured interviews were carried out with a total of 11 surgeons in BC. Interviews were transcribed for thematic analysis via interpretive description. Dominant themes were identified and agreed upon between the authors. RESULTS: Several barriers were identified, which include system and institution, communication and surgical workflow barriers. At the system and institutional level, there were difficulties accessing patient information and continuity of care. Themes in the communication included patient misconceptions about palliative care and communication challenges with consulting services. Surgical workflow barriers influenced the overall perceived role of surgeons when caring for patients with palliative care needs. CONCLUSION: Understanding surgeons' perspectives on barriers to palliative care is an important step in changing management. This can aid in the development of strategies that ease access to palliative care.

Co-Morbidities of Irritable Bowel Syndrome in a Racially and Ethnically Diverse Population.

Introduction: Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction (DGBI), and associated co-morbidities worsen quality of life. Research concerning IBS co-morbidities in different racial/ethnic groups is very sparse. This study aimed to determine the prevalence rates of co-morbidities and possible differences in a multiracial/ethnic IBS cohort. Methods: Based on ICD-9-coded IBS diagnosis, 740 outpatients (≥18 years) were included in this retrospective study at Boston Medical Center. Demographics and ICD-9-coded co-morbidities were extracted from electronic records. Descriptive statistics and multiple logistic regression were used for data analyses. Results: The most prevalent co-morbidities in this IBS cohort included gastroesophageal reflux disorder (GERD) (30%), depression (27%), anxiety (23%), (chronic obstructive pulmonary disease) COPD/asthma (16%), and obesity (10%). GERD was more prevalent in Hispanics and Blacks (p = 0.0005), and non-ulcer dyspepsia (NUD) was more prevalent in Blacks and Asians (p = 0.003). Higher rates of diabetes mellitus type 2 (DMT2) (p = 0.0003) and depression (p = 0.03), but not anxiety (p = 0.9), were present in Blacks and Hispanics. GERD was significantly associated with Hispanics (p = 0.003), dependent on age, overweight, and obesity. NUD was significantly associated with Blacks (p = 0.01) and Asians (p = 0.006), independent of sex, age, and BMI. Cancer of the thyroid, ovaries, and testis occurred at a five-fold higher rate than expected. Conclusions: Significant racial/ethnic differences exist for IBS co-morbidities in this study cohort, including depression, DMT2, GERD, and NUD. Certain cancers were found to be more frequent in this IBS sample as compared with the general population.

Efficacy and safety of oral anticoagulants according to kidney function among patients with atrial fibrillation.

BACKGROUND AND AIMS: Patients with severely reduced kidney function have been excluded from randomized controlled trials and data on the safety and efficacy of direct oral anticoagulants (DOACs) according to kidney function remain sparse. The aim was to evaluate the safety and efficacy of the DOACs across subgroups of kidney function. METHODS: Using multiple Danish nationwide registers and laboratory databases, we included patients initiated on oral anticoagulants (OACs) with atrial fibrillation and available creatinine level and followed patients for 2 years to evaluate occurrence of stroke/thromboembolism (TE) and major bleeding. RESULTS: Among 26 686 included patients, 3667 (13.7%) had an estimated glomerular filtration rate (eGFR) of 30-49 mL/min/1.73 m2 and 596 (2.2%) had an eGFR below 30 mL/min/1.73 m2. We found no evidence of differences regarding the risk of stroke/TE between the OACs (P-value interaction >0.05 for all). Apixaban was associated with a lower 2-year risk of major bleeding compared to vitamin K antagonists (VKA) [hazard ratio 0.79, 95% confidence interval (CI) 0.67-0.93], and the risk difference was significantly larger among patients with reduced kidney function (P-value interaction 0.018). Rivaroxaban was associated with a higher risk of bleeding compared to apixaban (hazard ratio 1.78, 95%CI 1.32-2.39) among patients with eGFR 30-49 mL/min/1.73 m2. CONCLUSIONS: Overall, we found no differences regarding the risk of stroke/TE, but apixaban was associated with a 21% lower relative risk of major bleeding compared to VKA. This risk reduction was even greater when comparing apixaban to VKA among patients with eGFR 15-30 mL/min/1.73 m2, and when comparing apixaban to dabigatran and rivaroxaban among patients with eGFR 30-49 mL/min/1.73 m2.

Examining the Intersection of Ethnoracial Disparities and HIV Status in Substance Use Risks among U.S. Adults.

Black/African American and Hispanic Americans experience significant HIV-related disparities. Substance use might be a contributing factor to these disparities, but there is limited research on this topic. This study investigated various substance use risks by HIV status and race/ethnicity (Black, Hispanic, White) among U.S. adults. We used data from the 2005-2019 National Survey on Drug Use and Health (N = 541,921). In each racial/ethnic group, the prevalence rates of past-year and past-month tobacco, alcohol, cannabis, and cocaine use, and past-year alcohol and illicit drug use disorders were estimated by HIV status. A series of logistic regressions with the interaction term of HIV x race/ethnicity were performed to examine race/ethnicity's moderating effect on the HIV-substance use associations, while controlling for sociodemographic factors and survey year. Moderation analysis showed that HIV status's association with the risks of past-year tobacco use (AOR = 1.67, 95% CI = 1.01-2.75), past-year cocaine use (AOR = 3.80, 95% CI = 1.91-7.57), past-month cocaine use (AOR = 5.34, 95% CI = 2.10-13.60), and past-year alcohol use disorder (AOR = 2.52, 95% CI = 1.29-4.92) differed significantly between Black and White adults. Between the Hispanic and White groups, HIV status's association with the risks of past-year alcohol use (AOR = 2.00, 95% CI = 1.09-3.69), past-year cocaine use (AOR = 2.40, 95% CI = 1.06-5.39), and past-month cocaine use (AOR = 3.69, 95% CI = 1.36-10.02) also differed significantly. It is well-established that individuals with HIV face an elevated risk of substance use. Our study added valuable insights by highlighting that this phenomenon is particularly more significant among Black and Hispanic adults for several substances when compared to White adults. Implications for practice are discussed.

Identification of small molecule inhibitors of G3BP-driven stress granule formation.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Vascular Reconstruction and Limb Loss in Military Tibial Artery Injuries.

BACKGROUND: Selective operative management of injuries to the tibial arteries is controversial, with the necessity of revascularization in the face of multiple tibial arteries debated. Tibial artery injuries are frequently encountered in military trauma, but revascularization practices and outcomes are poorly defined. We aimed to investigate associations between the number of injured vessels and reconstruction and limb loss rates in military casualties with tibial arterial trauma. METHODS: A US military database of lower extremity vascular injuries from Iraq and Afghanistan (2004-2012) was queried for limbs sustaining at least 1 tibial artery injury. Injury, intervention characteristics, and limb outcomes were analyzed by the number of tibial arteries injured (1, T1; 2, T2; 3, T3). RESULTS: Two hundred twenty one limbs were included (194 T1, 22 T2, 5 T3). The proportions with concomitant venous, orthopedic, nerve, or proximal arterial injuries were similar between groups. Arterial reconstruction (versus ligation) was performed in 29% of T1, 63% of T2, and universally in T3 limbs (P < 0.001). Arterial reconstruction was via vein graft (versus localized repair) in 62% of T1, 54% of T2, and 80% of T3 (P = 0.59). T3 received greater blood transfusion volume (P = 0.02), and fasciotomy was used universally (versus 34% T1 and 14% T2, P = 0.05). Amputation rates were 23% for T1, 26% for T2, and 60% for T3 (P = 0.16), and amputation was not significantly predicted by arterial ligation in T1 (P = 0.08) or T2 (P = 0.34) limbs. Limb infection was more common in T3 (80%) than in T1 (25%) or T2 (32%, P = 0.02), but other limb complication rates were similar. CONCLUSIONS: In this series of military lower extremity injuries, an increasing number of tibial arteries injured was associated with the increasing use of arterial reconstruction. Limbs with all 3 tibial arteries injured had high rates of complex vascular reconstruction and eventual amputation. Limb loss was not predicted by arterial ligation in 1-vessel and 2-vessel injuries, suggesting that selective reconstruction in these cases is advisable.

Discovery of KB-0742, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of CDK9 for MYC-Dependent Cancers.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

Curved adhesions mediate cell attachment to soft matrix fibres in three dimensions.

Integrin-mediated focal adhesions are the primary architectures that transmit forces between the extracellular matrix (ECM) and the actin cytoskeleton. Although focal adhesions are abundant on rigid and flat substrates that support high mechanical tensions, they are sparse in soft three-dimensional (3D) environments. Here we report curvature-dependent integrin-mediated adhesions called curved adhesions. Their formation is regulated by the membrane curvatures imposed by the topography of ECM protein fibres. Curved adhesions are mediated by integrin ɑvß5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin ß5 and a curvature-sensing protein, FCHo2. We find that curved adhesions are prevalent in physiological conditions, and disruption of curved adhesions inhibits the migration of some cancer cell lines in 3D fibre matrices. These findings provide a mechanism for cell anchorage to natural protein fibres and suggest that curved adhesions may serve as a potential therapeutic target.

Two Listeria monocytogenes outbreaks in a cancer centre: onsite food premises and their potential health risk to patients.

BACKGROUND: This report describes two L. monocytogenes outbreak investigations that occurred in March and September of 2018 and that linked illness to a food premises located in an Ontario cancer centre. The cancer centre serves patients from across the province. METHODS: In Ontario, local public health agencies follow up with all reported laboratory-confirmed cases of listeriosis to identify possible sources of disease acquisition and to carry out investigations, including at suspected food premises. The Canadian Food Inspection Agency (CFIA) is notified of any Listeria-positive food product collected in relation to a case. The CFIA traces Listeria-positive product through the food distribution system to identify the contamination source and ensure the implicated manufacturing facility implements corrective measures. RESULTS: Outbreaks one and two each involved three outbreak-confirmed listeriosis cases. All six cases were considered genetically related by whole genome sequencing (WGS). In both outbreaks, outbreak-confirmed cases reported consuming meals at a food premises located in a cancer centre (food premises A) before illness onset. Various open deli meat samples and, in outbreak two, environmental swabs (primarily from the meat slicer) collected from food premises A were genetically related to the outbreak-confirmed cases. Food premises A closed as a result of the investigations. CONCLUSIONS: When procuring on-site food premises, healthcare facilities and institutions serving individuals with immuno-compromising conditions should consider the potential health risk of foods available to their patient population.

A Surgical Technique for Closure of 10 mm and Larger Laparoscopic Port Fascial Defects Using a Graham's Nerve Hook.

Background and Objectives: In order to avoid potential complications from incisional hernias in patients undergoing laparoscopic or robotic procedures with 10 mm or larger ports, a surgeon closes the fascial defects using various techniques. We compared several different techniques of port site closure, which uses the open technique that can be performed with or without laparoscopic visualization. We modified the technique initially described by Dr. H. Aziz. We are introducing a new surgical technique to close the larger port site using Graham's nerve-hook. This new technique is easy to learn, replicate and implement for all body types. Methods: We use the commonly available Graham's nerve-hook and two S-retractors to visualize the entire layers of fascia and peritoneum and to pull up both layers to close the larger port site safely and securely with 0 polyglactin absorbable suture. We illustrated this new Lee's port site closure technique with eight separate drawings in this paper. Results: We performed 493 consecutive laparoscopic cases using this new technique. Four years follow up revealed only one incisional hernia using this technique. The patients are routinely followed in one month and six months and a year after the operation. However, not all of the patients are seen after six months unless there was a specific complaint. Conclusion: The new port site closure technique introduced in this paper is found to be easy to learn, fast, and very cost effective due to the reusable, commonly found S-retractors and Graham's nerve hook. After four years of consistent use, this new technique was found to be safe and effective in closure of 10 mm or larger port sites.

Diagnosis of Eosinophilic Esophagitis at the Time of Esophageal Food Impaction.

BACKGROUND: Esophageal food impactions (EFI) often precede a diagnosis of eosinophilic esophagitis (EOE). Current guidelines suggest obtaining esophageal biopsies upon suspicion of EOE, treating with proton pump inhibitor (PPI), and repeating esophagogastroduodenoscopy (EGD). This study was conducted to determine provider practice patterns with these mentioned recommendations at the time of EFI. METHODS: In this retrospective study, key outcomes were the proportion of patients who had EOE mucosal biopsies, EOE diagnosis, PPI initiation, and recommendations and completions of repeat EGD. Differences in outcomes among age, sex, race, off-hours time of procedure, and trainee involvement were examined. EOE diagnosis predictors were explored with logistic regression. RESULTS: Twenty-nine percent of the patients had esophageal biopsies taken at the time of index EGD (iEGD). Sixteen patients were diagnosed with EOE at the time of index EFI, while fourteen patients were diagnosed on subsequent EGDs. Among those diagnosed with EOE at iEGD, 94% were placed on PPI. Of patients with confirmed EOE on index biopsy, 63% of patients were recommended repeat EGD, of which 50% completed it within 90 days. Older age was protective of EOE diagnosis while no GERD history and endoscopist suspicion of EOE predicted diagnosis of EOE. CONCLUSIONS: Endoscopists uncommonly take biopsies at the time of EFI, which may delay diagnosis and treatment of EOE.

Arterial Access Complications Following Percutaneous Femoral Access in 24-Hour Resuscitative Endovascular Balloon Occlusion of the Aorta Survivors.

INTRODUCTION: With the use of resuscitative endovascular balloon occlusion of the aorta (REBOA) comes the potential for vascular access site complications (VASCs) and limb ischemic sequelae. We aimed to determine the prevalence of VASC and associated clinical and technical factors. METHODS: A retrospective cohort analysis of 24-h survivors undergoing percutaneous REBOA via the femoral artery in the American Association for the Surgery of Trauma Aortic Occlusion for Resuscitation in Trauma and Acute care surgery registry between Oct 2013 and Sep 2021 was performed. The primary outcome was VASC, defined as at least one of the following: hematoma, pseudoaneurysm, arteriovenous fistula, arterial stenosis, or the use of patch angioplasty for arterial closure. Associated clinical and procedural variables were examined. Data were analyzed using Fisher exact test, Mann-Whitney-U tests, and linear regression. RESULTS: There were 34 (7%) cases with VASC among 485 meeting inclusion criteria. Hematoma (40%) was the most common, followed by pseudoaneurysm (26%) and patch angioplasty (21%). No differences in demographics or injury/shock severity were noted between cases with and without VASC. The use of ultrasound (US) was protective (VASC, 35% versus no VASC, 51%; P = 0.05). The VASC rate in US cases was 12/242 (5%) versus 22/240 (9.2%) without US. Arterial sheath size >7 Fr was not associated with VASC. US use increased over time (R2 = 0.94, P < 0.001) with a stable rate of VASC (R2 = 0.78, P = 0.61). VASC were associated with limb ischemia (VASC, 15% versus no VASC, 4%; P = 0.006) and arterial bypass procedures (VASC 3% versus no VASC 0%; P < 0.001) but amputation was uncommon (VASC, 3% versus no VASC, 0.4%; P = 0.07). CONCLUSIONS: Percutaneous femoral REBOA had a 7% VASC rate which was stable over time. VASC are associated with limb ischemia but need for surgical intervention and/or amputation is rare. The use of US-guided access appears to be protective against VASC and is recommended for use in all percutaneous femoral REBOA procedures.

Intentional enterotomies: validation of a novel robotic surgery training exercise.

While laparoscopic simulation-based training is a well-established component of general surgery training, no such requirement or standardized curriculum exists for robotic surgery. Furthermore, there is a lack of high-fidelity electrocautery simulation training exercises in the literature. Using Messick's validity framework, we sought to determine the content, response process, internal content and construct validity of a novel inanimate tissue model that utilizes electrocautery for potential incorporation in such curricula. A multi-institutional, prospective study involving medical students (MS) and general surgery residents (PGY1-3) was conducted. Participants performed an exercise using a biotissue bowel model on the da Vinci Xi robotic console during which they created an enterotomy using electrocautery, followed by approximation with interrupted sutures. Participant performance was recorded and then scored by crowd-sourced assessors of technical skill, along with three of the authors. Construct validity was determined via difference in Global Evaluative Assessment of Robotic Skills (GEARS) score, time to completion, and total number of errors between the two cohorts. Upon completion of the exercise, participants were surveyed on their perception of the exercise and its impact on their robotic training to determine content validity. 31 participants were enrolled and separated into two cohorts: MS + PGY1 vs. PGY2-3. Time spent on the robotic trainer (0.8 vs. 8.13 h, p = 0.002), number of bedside robotic assists (5.7 vs. 14.8, p < 0.001), and number of robotic cases as primary surgeon (0.3 vs. 13.1, p < 0.001) were statistically significant between the two groups. Differences in GEARS scores (18.5 vs. 19.9, p = 0.001), time to completion (26.1 vs. 14.4 min, p < 0.001), and total errors (21.5 vs. 11.9, p = 0.018) between the groups were statistically significant as well. Of the 23 participants that completed the post-exercise survey, 87% and 91.3% reported improvement in robotic surgical ability and confidence, respectively. On a 10-point Likert scale, respondents rated the realism of the exercise 7.5, educational benefit 9.1, and effectiveness in teaching robotic skills 8.7. Controlling for the upfront investment of certain training materials, each exercise iteration cost ~ $30. This study confirmed the content, response process, internal structure and construct validity of a novel, high-fidelity and cost-effective inanimate tissue exercise which successfully incorporates electrocautery. Consideration should be given to its addition to robotic surgery training programs.

The Characteristics and Results of Endovascular Devices in Trauma (CREDiT) study: Multi-institutional results.

INTRODUCTION: Endovascular techniques are increasingly used to repair major traumatic vascular injuries, but most endovascular implants are not designed/approved for trauma-specific indications. No inventory guidelines exist for the devices used in these procedures. We aimed to describe the use and characteristics of endovascular implants used for repair of vascular injuries to allow for better inventory management. METHODS: This CREDiT study is a six-year retrospective cohort analysis of endovascular procedures performed for repair of traumatic arterial injuries at five participating US trauma centers. For each treated vessel, procedural and device details were recorded and outcomes assessed with the aim of defining the range of implants and sizes used for these interventions. RESULTS: A total of 94 cases were identified; 58 (61%) were descending thoracic aorta, 14 (15%) axillosubclavian, 5 carotid, 4 abdominal aortic, 4 common iliac, 7 femoropopliteal, and 1 renal. Vascular surgeons performed 54% of cases, trauma surgeons 17%, IR/CT Surgery 29%. Systemic heparin was administered in 68% and procedures were performed a median of 9 h after arrival (IQR 3-24 h). Primary arterial access was femoral in 93% of cases, 49% were bilateral. Brachial/radial access was used primarily in 6 cases, and secondary to femoral in 9. The most common implant was self-expanding stent graft; 18% used >1 stent. Implants ranged in diameter and length based on vessel size. Five of 94 implants underwent reintervention (1 open surgery) at a median of 4d postop (range 2-60d). Two occlusions and 1 stenosis were present at follow-up at a median of 1 month (range 0-72 m). CONCLUSIONS: Endovascular reconstruction of injured arteries requires a broad range of implant types, diameters, and lengths which should be readily available in trauma centers. Stent occlusions/stenoses are rare and can typically be managed by endovascular means.

Replication and innate immune responses of two chikungunya virus genotypes in human monocyte-derived macrophages.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus, which causes epidemics of fever, joint pain and rash. There are three genotypes: West African, East/Central/South/Africa (ECSA) and Asian, with the latter two predominant globally. Genotype-specific differences in clinical presentations, virulence and immunopathology have been described. Macrophages are key cells in immune responses against CHIKV. Circulating blood monocytes enter tissue to differentiate into monocyte-derived macrophages (MDMs) in response to CHIKV infection at key replication sites such as lymphoid organs and joints. This study analyses differences in replication and induced immune mediators following infection of MDMs with Asian and ECSA CHIKV genotypes. Primary human MDMs were derived from residual blood donations. Replication of Asian (MY/06/37348) or ECSA (MY/08/065) genotype strains of CHIKV in MDMs was measured by plaque assay. Nineteen immune mediators were measured in infected cell supernatants using multiplexed immunoassay or ELISA. MY/08/065 showed significantly higher viral replication at 24 h post-infection (h p.i.) but induced significantly lower expression of proinflammatory cytokines (CCL-2, CCL-3, CCL-4, RANTES and CXCL-10) and the anti-inflammatory IL-1Ra compared to MY/06/37348. No differences were seen at later time points up to 72 h p.i. During early infection, MY/08/065 induced lower proinflammatory immune responses in MDMs. In vivo, this may lead to poorer initial control of viral infection, facilitating CHIKV replication and dissemination to other sites such as joints. This may explain the consistent past findings that the ECSA genotype is associated with greater viremia and severity of symptoms than the Asian genotype. Knowledge of CHIKV genotype-specific immunopathogenic mechanisms in human MDMs is important in understanding of clinical epidemiology, biomarkers and therapeutics in areas with co-circulation of different genotypes.

Curved adhesions mediate cell attachment to soft matrix fibres in 3D.

Mammalian cells adhere to the extracellular matrix (ECM) and sense mechanical cues through integrin-mediated adhesions 1, 2 . Focal adhesions and related structures are the primary architectures that transmit forces between the ECM and the actin cytoskeleton. Although focal adhesions are abundant when cells are cultured on rigid substrates, they are sparse in soft environments that cannot support high mechanical tensions 3 . Here, we report a new class of integrin-mediated adhesions, curved adhesions, whose formation is regulated by membrane curvature instead of mechanical tension. In soft matrices made of protein fibres, curved adhesions are induced by membrane curvatures imposed by the fibre geometry. Curved adhesions are mediated by integrin ɑVß5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin ß5 and a curvature-sensing protein FCHo2. We find that curved adhesions are prevalent in physiologically relevant environments. Disruption of curved adhesions by knocking down integrin ß5 or FCHo2 abolishes the migration of multiple cancer cell lines in 3D matrices. These findings provide a mechanism of cell anchorage to natural protein fibres that are too soft to support the formation of focal adhesions. Given their functional importance for 3D cell migration, curved adhesions may serve as a therapeutic target for future development.